Effect of oral or injectable supplementation with cobalamin in dogs with hypocobalaminemia caused by chronic enteropathy or exocrine pancreatic insufficiency

Abstract Background Recent studies have shown similar efficacy of oral supplementation of cobalamin compared to injectable supplementation in dogs, but few prospective, randomized studies have been published. Objectives To evaluate efficacy of oral or injectable supplementation with cobalamin in normalizing serum cobalamin and methylmalonic acid (MMA) concentrations in dogs with hypocobalaminemia caused by either chronic enteropathy (CE) or exocrine pancreatic insufficiency (EPI). Animals Forty‐six client owned dogs with hypocobalaminemia. Methods Prospective randomized clinical trial. Dogs were divided into 2 groups (CE or EPI), and randomized to receive oral or injectable supplementation of cobalamin. Each dog had 3 visits and serum cobalamin and MMA concentrations were measured at each visit. Results In dogs with CE, serum cobalamin concentrations increased with oral (P = .02; median 149 [range 149‐231] to 733 [166‐1467] ng/L, median difference 552 [95% CI: 181‐899] ng/L) or injectable (P < .01; 168 [149‐233] to 563 [234‐965] ng/L, 367 [187‐623] ng/L) supplementation. In dogs with EPI, serum cobalamin concentrations increased with oral (P = .01; 162 [149‐214] to 919 [643‐3863] ng/L, 705 [503‐3356] ng/L) or injectable (P = .01; 177 [149‐217] to 390 [243‐907] ng/L, 192 [89‐361] ng/L) supplementation. Serum MMA concentrations decreased with oral or injectable supplementation in dogs with CE, but only with oral supplementation in dogs with EPI. Conclusions and Clinical Importance Oral supplementation is an alternative for cobalamin supplementation in dogs with hypocobalaminemia caused by CE or EPI.

K E Y W O R D S alternative route, cobalamin deficiency, methylmalonic acid, small intestinal disease

| INTRODUCTION
Cobalamin is a required intracellular cofactor for 2 enzymes that are essential for maintaining cellular functions in mammals. [1][2][3] Cobalamin that dogs obtain from their diet must combine with intrinsic factor (IF) before absorption in the ileum as a complex by specific receptors. 1,4 Because the exocrine pancreas is the main source for IF in dogs, 5,6 exocrine pancreatic insufficiency (EPI) is a common cause of hypocobalaminemia in dogs. 7 Another common cause of hypocobalaminemia in dogs is chronic small intestinal disease where damaged ileal mucosa results in reduced cobalamin receptor expression with subsequently diminished cobalamin absorption. 8 Other less common causes include congenital disorders of the cobalamin receptor and small intestinal dysbiosis. 1,9 Hypocobalaminemia in dogs can manifest as lethargy, inappetence, vomiting, diarrhea, weight loss, anemia, and failure to thrive in puppies. 1,2,10 It is important that clinicians evaluate cobalamin status in dogs with chronic enteropathy (CE) or EPI because hypocobalaminemia is a negative prognostic factor in these diseases. 7,8 Serum cobalamin concentration is commonly measured to evaluate cobalamin status; however, it is not an accurate marker for cobalamin status on a cellular level. Methylmalonic acid (MMA) is a better marker for cellular cobalamin status because MMA accumulates when intracellular cobalamin level is abnormally low. 1 Serum MMA concentration is a marker for cellular cobalamin status [11][12][13] ; however, serum MMA concentration can also be affected by renal diseases or certain enzyme defects. 14,15 Both hypocobalaminemia and cobalamin deficiency are used interchangeably to describe low serum cobalamin concentrations. However, a serum cobalamin concentration below the lower limit of the reference interval (RI) should be referred to as hypocobalaminemia (≤250 ng/L) and hypocobalaminemia with an increased serum MMA concentration as cobalamin deficiency. 11,13 Many clinicians recommend supplementation regardless of the underlying etiology when serum cobalamin concentration is in the lower part of the RI (<400 ng/L at our institution). 16 While subcutaneous administration of cobalamin is the mainstay of supplementation, recent oral supplementation is reported to be similarly efficacious in normalizing serum or cellular cobalamin concentration in dogs with CE or hereditary cobalamin malabsorption. [17][18][19][20] Among these studies, there are 2 prospective studies in dogs with CE evaluating the efficacy of oral supplementation of cobalamin. 18,20 Also, only 1 recent pilot study exists evaluating the efficacy of oral supplementation of cobalamin in dogs with EPI. 21 The aim of this study was to evaluate the efficacy of oral or injectable supplementation with cobalamin in normalizing serum cobalamin and MMA concentrations in dogs with hypocobalaminemia caused by either CE or EPI. We hypothesized that both oral and injectable supplementation with cobalamin would have efficacy in normalizing serum cobalamin and MMA concentrations in dogs with hypocobalaminemia caused by CE or EPI.

| Animals
Client owned dogs were considered eligible for enrollment into this study based on the results of measurement of serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin like immunoreactivity (cTLI) submitted to the Gastrointestinal Laboratory (GI Lab) at Texas A&M University. Veterinarians were instructed to collect all venous blood samples after withholding food for at least 10 to 12 hours. Inclusion criteria consisted of a serum cobalamin concentration below the lower limit of the RI (≤250 ng/L), a cPLI in the RI (≤200 μg/L), and a cTLI in the RI (5.7-45.2 μg/L) or a cTLI below 2.5 μg/L, consistent with EPI. Dogs with any history of cobalamin supplementation or systemic, extragastrointestinal disease were excluded. Treatment of enrolled dogs beyond cobalamin supplementation was at the discretion of the attending veterinarian. Also, dogs could be fed any diet during the study period. Dogs were enrolled based on a history of signs of chronic GI disease for more than 3 weeks duration and results of blood work performed at baseline to exclude extra-intestinal causes of the signs of GI disease. The blood work included CBC, serum chemistry profile, and the repeat GI panel (serum cobalamin, folate, cPLI, and cTLI). Signed informed consent was obtained from each owner before enrollment. The animal use protocol for this study was approved by the Animal Care and Use Committee of our institution (IACUC 2015-0286, IACUC 2018-0347).

| Study design and cobalamin supplementation
This project was performed as a prospective randomized clinical trial.
The enrolled dogs were classified into 2 different groups (CE or EPI) based on the cTLI results, with each dog then randomly assigned to 1 of 3 treatment groups (injectable supplementation of cobalamin, oral supplementation of cobalamin, or oral supplementation of cobalamin with folate), regardless of serum folate concentration at baseline. Randomization of each dog was achieved by following the ordered numbers prepared using the block randomization method performed by Nutramax Laboratories (Lancaster, South Carolina). Dogs in the injectable treatment group received weekly subcutaneous administration of cyanocobalamin (Vitamin B12 injection 1000 μg/mL, SPARHAWK Laboratories, Lenexa, Kansas) for 6 weeks, with an additional subcutaneous injection 4 weeks later for a total of 7 subcutaneous injections.
Every subcutaneous injection was administered by a veterinary professional in a clinic setting. Dogs in the oral treatment groups (cobalamin only or cobalamin with folate) received daily oral supplementation using chews containing either cyanocobalamin or cyanocobalamin and folate, respectively, for a total of 12 weeks. Both chews were manufactured by Nutramax Laboratories (Lancaster, South Carolina). The dosage of cobalamin supplementation in each treatment group was determined based on the body weight of each dog (Table 1). Each dog had 3 visits with their primary veterinarian for baseline and 2 recheck visits during the study. During each visit, a pertinent history was obtained, a standard physical examination was performed, and a venous blood sample was drawn after withholding food for at least 10 to 12 hours. The first recheck was scheduled 6 weeks after initiation of cobalamin supplementation (week 7), and the second recheck was performed 1 week after cessation of supplementation (week 11 for injectable treatment group and week 13 for both oral treatment groups).

| Blood sample processing
After collection of a venous blood sample (about 5 mL), approximately 1 mL of whole blood was aliquoted into EDTA anticoagulant containing blood tubes, with the remaining whole blood was centrifuged to obtain serum. The separated serum (at least 1.5 mL) was aliquoted into plain red top tubes, and all samples were shipped to the GI Lab overnight on an icepack. When the blood samples were not shipped within the same business day, the blood tubes refrigerated (3-5 C) overnight or over the weekend up to 48 hours. The EDTA containing blood tubes were submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for a CBC. The serum samples were aliquoted into several sterile plastic tubes and evaluated for a chemistry profile, GI panel, and MMA concentrations. The laboratory's RI for serum folate and TLI in dogs are 7.7 to 24.4 and 5.5 to 45.2 μg/L, respectively. Serum MMA concentration was measured by the stable isotope dilution gas chromatography-mass spectrometry method as described in previous studies, 11,22 and the RI for serum MMA in dogs was previously determined to 415 to 1193 nmol/L. 11 Serum cPLI was measured using a commercially available ELISA kit (Spec cPL, IDEXX Laboratories, Westbrook, Maine) in the GI Lab, and the RI for cPLI in dogs is ≤200 μg/L at this laboratory.

| Daily online questionnaire for owners
The owners of all enrolled dogs were asked to fill out a daily online questionnaire during the entire study period. The questionnaire consisted of several questions regarding ease of administration (for oral treatments only), appetite, vomiting, diarrhea, or any other adverse effects related to the cobalamin supplementation.

| Statistical analysis
Serum cobalamin, folate, and MMA concentrations were statistically evaluated. Because none of the data were normally distributed, the Friedman tests followed by Dunn's multiple comparison tests were performed to compare the data (serum cobalamin, folate, and MMA concentrations) between different time points (baseline, first and second recheck) in each treatment group. Because no significant change of serum folate was identified in either oral treatment group (cobalamin only or cobalamin with folate), the data from both oral treatment groups were combined to increase statistical power. The final statistical analysis was performed using 2 treatment groups (combined oral group and injectable group) and shown in the results.
Analysis was performed using a commercially available statistical software package (GraphPad Prism 9.2), and statistical significance was set at P < .05.  In dogs with EPI, the most common chief complaints at baseline were also chronic diarrhea (

| Complete blood counts and chemistry profiles
Complete blood count and chemistry profiles were performed at 3 time points for each dog. The results are shown in Tables 2 and 3, and more detailed explanations are described in Data S1.
F I G U R E 1 This flowchart shows the study process from initial evaluation to completion, including the number of dogs and reasons for exclusion at baseline and during the study

| Serum cPLI and cTLI concentrations
In the CE group, all dogs in both the oral and the injectable treatment groups had normal cPLI and cTLI concentrations. In the EPI group, all dogs in either the oral or the injectable treatment groups had normal serum cPLI concentrations. Serum cTLI concentrations were consistent with EPI in all dogs of the EPI group.

| Online daily questionnaire for owners
The results of the questionnaire are summarized in Table 5, and more detailed explanations are described in Data S1.

| DISCUSSION
The results of this prospective, randomized clinical trial indicate that oral or injectable supplementation of cobalamin is efficacious in nor- There was a significant increase of serum cobalamin concentrations after treatment suggesting that oral supplementation of cobalamin is a potential alternative to injectable treatment in dogs with EPI.
Limitations of this study were the retrospective design and a lack of serum MMA concentrations for most dogs (ie, serum MMA was only available for 4 dogs).
Our results in dogs with CE identified that the median and median

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.